Bio-Rad Affi-Gel Protein A Media - Manual (Page 7)

3. Apply diluted sample to the column.
4. Wash the column with 15 bed volumes of binding buffer.
5. Elute the IgG with 5 bed volumes of elution buffer. Elute col-
umn with an additional 10 volumes of buffer to insure total
removal of IgG. Prolonged exposure to acid pH should be avoid-
ed. Neutralize eluate immediately after elution. For example,
for a 1 ml column, collect eluate in a tube containing 1.6 ml of
1 M Tris HCl, pH 9. Sample pH under these conditions is 6 to
8. Alternatively, eluate can be dialyzed against the buffer of
choice, or desalted over Bio-Gel
® P6DG desalting gel. See bul-
letin 2068 for desalting protocols.
6. Wash the column with 5 bed volumes of regeneration buffer.
7. Store the PBS containing 0.05% sodium azide.
Note: For all sample sizes, short columns (10-15 cm or shorter)
are recommended. The better linear flow rates obtained in short
columns facilitate the washing and regeneration steps.
Answers to Common Questions
1. Regeneration of column:
Affi-Gel protein A agarose can be regenerated 10-12 times with
the regeneration buffer supplied. We strongly recommend regen-
erating the column each time it is used to increase the lifetime
of the gel to insure consistent separations from run to run. Cross-
over contamination of IgG from application to application will
also be eliminated.
2. Sensitivity of antibodies to low pH:
There are a few antibodies that are inactivated by low pH.
Inactivation can be avoided by collecting the fraction eluted
into a concentrated neutral buffer. In cases of extreme sensitiv-
ity, many immunoglobulins can be eluted at pH 4-6 by bringing
up the pH of the elution buffer with 10 N NaOH.
3. Flow rate of Affi-Gel protein A agarose:
5
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